Electron
MicroscopyIndications for performing this test: Because viral particles are too small to be visualized using light microscopy, other techniques are necessary to visualize them or confirm their presence. The following tools can be used to identify viral particles or viral antigen in fresh feces: electron microscopy, latex agglutination, ELISA. Remember that you have to be aware of what kind of detection systems are available for each virus and the relative sensitivities and specificies of any tests which are offered. There are also difference between animal species (e.g. a test for fecal detection of a rotavirus may be useful in foals, but less so in calves).
Although it sounds difficult, this is a common test run by diagnostic labs to detect a number of important enteric pathogens. The technique allows visualization and identification of many viruses. The procedure requires that feces are clarified by low speed centrifugation. The resulting supernatant is then subjected to ultracentrifugation (often through a sucrose cushion) to pellet the virions. The specimens are then stained negatively with phosphotungstate and scanned by electron microscopy. The negative staining procedure involves the addition of a solution of potassium phosphotungstate, which is electron dense, to a virus suspension on a coated specimen grid. The solution surrounds and fills the interstices in the surface of virions, giving a negative image of the virion to show details not previously seen. The advantages of this technique are that it allows visualization and identification of many viruses, and some non-cultivatable viruses can be found. The disadvantages include low sensitivity, the need for skilled personnel, time for the test to be run, and the need for expensive equipment.
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Detection of antibody to bovine
coronavirus by immunogold staining. Transmission electron microscopy. Source: A. Serafino, S. Favale, P.M. Cereda, A. Cristallo, M. Battaglia - Immunomicroscopia elettronica per la dimostrazione di anticorpi diretti contro i coronavirus. Giornata di Studio Istituto Medicina Sperimentale. Rome, 12 June 1995. Proceedings, p.45-47. S.T.A.R, 1996. |
This technique is the simplest of all immunoassays. It involves the agglutination by antigen of small latex beads previously coated with antiviral antibody. The test can be read by eye within a minute or two. The disadvantages are low sensitivity and and low specificity, so false negatives occur unless a large number of virions are present. Because of these facts, this assay for antigen tends to be restricted to examination of feces. Unfortunately, false positives occur quite commonly with fecal specimens.
ELISAs (enzyme linked immunosorbent assays) can be designed to detect viral antigens or antibodies. These tests are potentially very sensitive. Less than 1 nanogram of viral antigens per ml can be detected in specimens taken directly from the animal. There are a number of pathogens for which fecal ELISA's are available, including the Idexx SNAP test for parvovirus in dogs and a test for rotavirus in foals. The Washington Animal Diagnostic Disease Laboratory (WADDL) is still using electron microscopy to detect fecal rotavirus and coronavirus in agricultural animals because it has thus far proven to be more sensitive and reliable.
